Yeast Cell Surface Engineering of a Nicotinamide Riboside Kinase for the Production of β-Nicotinamide Mononucleotide via Whole- Cell Catalysis

fi-Nicotinamide mononucleotide (NMN) has been widely used as a nutraceutical for self-medication. The one-step conversion of nicotinamide riboside (NR) to fi-NMN has been considered to be the most promising synthetic route for fi-NMN. Here, human nicotinamide riboside kinase 2 (NRK-2) was functionally displayed on the cell surface of Saccharomyces cerevisiae EBY100, forming a whole-cell biocatalyst (Whole cell NRK-2). Whole-cell NRK-2 could convert nicotinamide riboside (NR) to fi-NMN efficiently in the presence of ATP and Mg2+, with a maximal activity of 64 IU/g (dry weight) and a Km of 3.5 mu M, similar to that of free NRK-2 reported previously. To get the best reaction conditions for fi- NMN synthesis, the amounts of NR, ATP, and Mg2+ used, pH, and temperature for the synthetic reaction were optimized. Using Whole-cell NRK-2 as the catalyst under the optimized conditions, fi-NMN synthesized from NR reached a maximal conversion rate of 98.2%, corresponding to 12.6 g/L of fi-NMN in the reaction mixture, which was much higher than those of synthetic processes reported. Additionally, Whole-cell NRK-2 had good pH stability and thermostability, required no complicated treatments before or after use, and could be reused in sequential production. Therefore, this study provided a safe, stable, highly effective, and low-cost biocatalyst for the preparation of fi-NMN, which has great potential in industrial production.